1. Field of the Invention
This invention relates to nonnucleoside-containing, fluorescence-tagged, phosphorus reagents, which are useful in the preparation of 5'-tagged oligonucleotides. A class of 5'-fluorescence-tagged oligonucleotides is also disclosed as part of this invention.
2. Summary of the Background
Deoxyribonucleic acid (DNA) is the molecule that stores the information needed to direct all processes in living systems. It is a polymer composed of four mononucleotide subunits linked by phosphodiester bonds. Naturally occurring DNA is usually found in a double-stranded form with two complimentary linear polymers held together by hydrogen bonds. Double-stranded DNA can be dissociated into single strands of DNA and, conversely, complimentary single-stranded DNA will associate to form double-stranded DNA.
Although the terms "DNA" and "oligonucleotide" are often used interchangeably, "DNA" is used herein to refer to large (&gt;100 nucleotides long) or naturally occurring molecules, especially those being subjected to various assays. "Oligonucleotide" is used herein to refer to pieces of single-stranded DNA that are small enough to be made by current, practical chemical synthesis (&lt;100 nucleotides long). The distinction between the terms "DNA" and "oligonucleotide," however, is recognized to be somewhat artificial. DNA can be broken into well-defined pieces that are small enough to be considered as oligonucleotides, and chemically synthesized oligonucleotides can be joined enzymatically to make double-stranded polymers large enough to be called DNA and to direct life processes.
The ability to introduce reporters into oligonucleotides and DNA is, in part, responsible for the recent explosive growth in the field of molecular biology. A reporter can be defined as a chemical group that has a physical or chemical characteristic readily measurable or detectable by appropriate physical or chemical detector systems or procedures. Ready detectability can be provided by such characteristics as color change, luminescence, fluorescence, and radioactivity or it may be provided by the ability of the reporter to serve as a ligand recognition site to form specific ligand-ligand complexes in which the second ligand contains a group detectable by conventional (e.g., colorimetric, spectrophotometric, fluorometric or radioactive) detection procedures. The ligand-ligand complexes can be in the form of protein-ligand, enzyme-substrate, antibody-antigen, carbohydrate-lectin, protein-cofactor, protein-effector, nucleic acid-nucleic acid, or nucleic acid-ligand complexes. The complex formed between biotin and avidin is an example of such a ligand-ligand complex.
Although high specific activity .sup.32 P has generally been used to tag oligonucleotides as well as DNA for a variety of applications, the use of this radioisotope is problematic from both a logistical and a health standpoint. The short half-life of .sup.32 P necessitates the anticipation of reagent requirements several days in advance and prompt use of such a reagent. Once .sup.32 P-tagged DNA sequencing fragments have been generated, they are prone to self-destruction and must be immediately subjected to electrophoretic analysis. Subsequent autoradiography required for visualization of the labeled DNA fragments in the electrophoretic gel is a slow process (overnight exposures are common). Finally, possible health risks are associated with the use and disposal of such potent radioisotopes.
To address these problems, replacement of .sup.32 P/autoradiography with alternative, nonradioisotopic reporter/detection systems has been considered. New reporter/detection systems must be exceptionally sensitive to replace .sup.32 P. In one sense, DNA can be its own "reporter" because it can be detected by ultraviolet (UV) light absorption. Many important assays, however, require that DNA be detected at concentrations many orders of magnitude below concentrations at which DNA can be detected by UV absorbance. DNA sequencing, for example, requires reporter/detection systems that can detect 10.sup.-16 mole (or about 10.sup.6 molecules) of DNA. Therefore, practical non-isotopic reporter/detection systems must offer sensitivity at least comparable to that of .sup.32 P. Hereafter, the term "reporter" shall refer only to chemical groups capable of replacing high specific activity .sup.32 P.
Prober, et al., Science 238, 336-41 (1987) and Smith, et al., Nature 321, 674-79 (1986), have shown that, in conjuction with an argon laser and filtered photomultiplier tube detection system, certain fluorescent dyes can replace .sup.32 P as reporters for DNA sequencing. To achieve the required sensitivity, these dyes were carefully selected for their strong absorption at the wavelength of the argon laser, their high quantum efficiency of fluorescent emission, and the ability to distinguish their fluorescent emission from background signals.
The ability to introduce readily detected reporters at a specific site in DNA is absolutely critical to many methods of analyzing DNA. For example, all currently known methods for sequencing DNA require that several hundred different oligonucleotides be separated by gel electrophoresis. (About 10.sup.-15 to 10.sup.-16 mole of each oligonucleotide is generally present.) Therefore, it is critical that the reporter does not complicate separation by gel electrophoresis. .sup.32 P is an ideal reporter in this respect because substituting .sup.32 P for nonradioactive .sup.31 P has no effect on gel electrophoresis. When a nonradioactive reporter, such as a fluorescent dye, is attached to an oligonucleotide, the electrophoretic mobility of that oligonucleotide changes. If only a single reporter is attached to the oligonucleotide at a precisely defined location, such changes are uniform and tolerable. If, however, a variable number of reporters are attached or if a single reporter is attached to a variety of positions, electrophoretic analysis becomes impossible.
DNA amplification by the polymerase chain reaction (PCR) is another technique for analyzing DNA that requires separation by gel electrophoresis. Preferably, oligonucleotides used in this method will also have a non-isotopic reporter at a single location.
Although several methods of non-site-specific enzymatic tagging of DNA are known, only one type of site-specific tagging with non-isotopic reporters is known. The enzyme, terminal transferase, is capable of adding a variety of modified and/or tagged nucleotides to the 3'-end of an oligonucleotide. This enzyme affects single-site tagging only when the 3'-hydroxyl group of the modified and/or tagged nucleotide is removed or changed. Unfortunately, DNA tagged by this method cannot be used in many enzymatic assays. DNA sequencing and amplification, for example, require that the tagged oligonucleotides used in these assays have a normal hydroxyl group at the 3'-end.
Many chemical methods for tagging DNA have been developed, but most of these involve non-site-specific reactions, thereby producing tagged DNA that is not suitable for analysis by gel electrophoresis. Site-specific tagged oligonucleotides have been prepared by total, i.e., chemical, synthesis. With one exception, this has always been done by synthesizing an oligonucleotide possessing an added group with unusual reactivity, e.g., an aliphatic amino group or a thiol. In this approach, the added amino or thiol groups have either replaced the 5'-hydroxyl group or have been added to the 5'-hydroxyl group by means of a linker or have been added to the base by means of a linker. This site-specific tagging process comprises: (i) preparation of a monomeric nucleotide reagent that contains a protected form of the unusually reactive group; (ii) chemical synthesis and purification of the desired oligonucleotides with the unusually reactive group, usually with concomitant deprotection of the unusually reactive group; and (iii) selective attachment of a fluorescent dye (or other reporter) to the unusually reactive group.
Examples of this and related approaches have been disclosed by Draper, et al., Biochemistry 19, 1774-81 (1980); Smith, DE No. 3,446,635 Al (1985); Smith, et al., Nucleic Acids Res. 13, 2399-2412 (1985); Coull, et al., Tetrahedron Lett. 27, 3991-94 (1986); Sproat, et al., Nucleic Acids Res. 15, 4837-48 (1987); Sproat, et al., Nucleic Acids Res. 15, 6181-96 (1987); Tanaka, et al., Tetrahedron Lett. 28, 2611-14 (1987); Tanaka, et al., Nucleic Acids Res. 15, 6209-24 (1987); Agrawal, et al., Nucleic Acids Res. 14, 6227-45 (1986); Connolly, Nucleic Acids Res. 15, 3131-39 (1987); Connolly, et al., Nucleic Acids Res. 13, 4485-4502 (1985); and Sinha, et al., Nucleic Acids Res. 16, 2659-69 (1988).
Totally synthetic site-specific tagging approaches present several problems in the synthesis of tagged oligonucleotides.
First, it is a multi-step procedure involving synthesis and purification of a modified oligonucleotide, addition of the reporter to the reactive group of this modified oligonucleotide, and a final purification.
Second, both DNA sequencing and DNA amplification require that the tagged oligonucleotide be a substrate for a DNA polymerase. Because these polymerases catalyze reactions at the 3'-end of the oligonucleotide, the 5'-end of the oligonucleotide is the preferred site for attaching non-isotopic reporters. When the unusually reactive group is attached to or incorporated within a nucleotide, this approach lacks versatility. The 5'-nucleotide can be dA, dT, dC or dG; therefore, four appropriate reagents are needed for incorporating an unusually reactive group along with the desired 5'-nucleotide. Because these reagents are typically air- and moisture-sensitive and have a limited shelf-life, the need to stock a set of at least four reagents is burdensome.
Third, if a linking group is used to introduce the unusually reactive functional group onto the 5'-position, additional problems arise. It is frequently difficult to determine whether the unusually reactive group has been successfully linked to a synthetic oligonucleotide. Because the reagents used to attach the unusually reactive group to the oligonucleotide have a limited shelf-life, failure to incorporate the desired reporter is common.
Fourth, when problems are encountered, it is usually difficult to determine which step failed.
Fifth, large excesses of the reporter are generally used for successful coupling to the unusually reactive group. This both wastes the reporter and complicates the purification of the oligonucleotide.
In the one exception to the totally synthetic site-specific tagging approaches described above, Prober, et al., EP-A No. 252,683 (1988), have outlined a more direct and reliable method for synthesizing fluorescence-tagged oligonucleotides for DNA sequencing. An unusually reactive functional group was not used in this approach. Instead, a fluorescent reporter was attached directly to a nucleotide before the nucleotide was incorporated into the desired oligonucleotide.
The principal disadvantage of this method is that it relies on attachment of the reporter to a specific nucleotide and therefore lacks versatility. The resulting fluorescence-tagged oligonucleotide was used in a DNA sequencing system that calls for four distinguishable fluorescent dyes. Complete versatility would require a set of 16 different fluorescence-tagged nucleotide reagents suitable for the synthesis of oligonucleotides. The reagents are also air- and moisture-sensitive and have a limited shelf-life.
The purpose of the present invention is to overcome the problems encountered in the prior art by providing nonnucleoside-containing, fluorescence-tagged, phosphorus reagents to produce 5'-tagged oligonucleotides. The reagents disclosed in the present invention are easier to prepare and are more versatile than are the compounds found in Prober, et al., EP-A No. 252,683. The presence of the reporters of the present invention in the resulting oligonucleotides does not interfere with analysis by gel electrophoresis or with use in DNA sequencing or DNA amplification. Fewer steps are required and the chances for error or confusion have been reduced when these reporters are used.
The 5'-tagged oligonucleotides of the present invention can be used as labeled primers for automated DNA sequencing and for DNA amplification by the polymerase chain reaction (PCR).